Henrike Brundiek

Creation of a Lipase Highly Selective for trans Fatty Acids by Protein Engineering

Sorting out: Protein engineering of lipase CAL-A led to the discovery of mutants with excellent chemoselectivity for the removal of trans and saturated fatty acids from partially hydrogenated vegetable oil. These fatty acids, identified as a major risk factor for human health, can now be removed by enzyme catalysis.

Enhancing the Acyltransferase Activity of Candida antarctica Lipase A by Rational Design.

A few lipases, such as Candida antarctica lipase A (CAL‐A), are known to possess acyltransferase activity. This enables the enzyme to synthesize fatty acid esters from natural oils and alcohols even in the presence of bulk water. Unfortunately, fatty acids are still formed in these reactions as undesired side‐products. To reduce the amount of fatty acids, several CAL‐A variants were rationally designed based on its crystal structure. These variants were expressed in Escherichia coli and Pichia pastoris, purified, and their acyltransferase/hydrolase activities were investigated by various biocatalytic approaches. Among the investigated variants, mutant Asp122Leu showed a significant decrease in the hydrolytic activity, thus reducing the side‐product yield during acylation. As desired, this variant retained wild‐type process‐relevant features like pH profile and thermostability.

Engineering and application of enzymes for lipid modification, an update

This review first provides a brief introduction into the most important tools and strategies for protein engineering (i.e. directed evolution and rational protein design combined with high-throughput screening methods) followed by examples from literature, in which enzymes have been optimized for biocatalytic applications. This covers engineered lipases with altered fatty acid chain length selectivity, fatty acid specificity and improved performance in esterification reactions. Furthermore, recent achievements reported for phospholipases, lipoxygenases, P450 monooxygenases, decarboxylating enzymes, fatty acid hydratases and the use of enzymes in cascade reactions are treated.

Protein engineering of enzymes involved in lipid modification

Abstract This chapter provides an overview of the most important protein engineering tools and strategies applied to enzymes involved in lipid modification. Thus, application of several methodologies is discussed, including directed evolution, rational protein design, construction of chimeric enzymes, and de novo design, together with the utility of bioinformatic tools along the protein engineering process. Additionally, different approaches for the creation of semirational libraries and the potential use of different high-throughput screenings for the detection of improved protein variants are described. The examples provided cover the recent achievements in the utilization of these techniques on a broad variety of enzymes. This comprises not only engineered lipases with altered fatty acid chain length selectivity, fatty acid specificity, and improved performance in esterification reactions, but also successfully altered phospholipases, lipoxygenases, P450 monooxygenases, decarboxylating enzymes, and fatty acid hydratases.

Alteration of Chain Length Selectivity of Candida antarctica Lipase A by Semi-Rational Design for the Enrichment of Erucic and Gondoic Fatty Acids

Abstract Biotechnological strategies using renewable materials as starting substrates are a promising alternative to traditional oleochemical processes for the isolation of different fatty acids. Among them, long chain mono‐unsaturated fatty acids are especially interesting in industrial lipid modification, since they are precursors of several economically relevant products, including detergents, plastics and lubricants. Therefore, the aim of this study was to develop an enzymatic method in order to increase the percentage of long chain mono‐unsaturated fatty acids from Camelina and Crambe oil ethyl ester derivatives, by using selective lipases. Specifically, the focus was on the enrichment of gondoic (C20:1 cisΔ11) and erucic acid (C22:1 cisΔ13) from Camelina and Crambe oil derivatives, respectively. The pursuit of this goal entailed several steps, including: (i) the choice of a suitable lipase scaffold to serve as a protein engineering template (Candida antarctica lipase A); (ii) the identification of potential amino acid targets to disrupt the binding tunnel at the adequate location; (iii) the design, creation and high‐throughput screening of lipase mutant libraries; (iv) the study of the selectivity towards different chain length p‐nitrophenyl fatty acid esters of the best hits found, as well as the analysis of the contribution of each amino acid change and the outcome of combining several of the aforementioned residue alterations and, finally, (v) the selection and application of the most promising candidates for the fatty acid enrichment biocatalysis. As a result, enrichment of C22:1 from Crambe ethyl esters was achieved either, in the free fatty acid fraction (wt, 78%) or in the esterified fraction (variants V1, 77%; V9, 78% and V19, 74%). Concerning the enrichment of C20:1 when Camelina oil ethyl esters were used as substrate, the best variant was the single mutant V290W, which doubled its content in the esterified fraction from approximately 15% to 34%. A moderately lower increase was achieved by V9 and its two derived triple mutant variants V19 and V20 (27%).